Bottom Line:
Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway.We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation.In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context.

ABSTRACTRaltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation 'freezes' IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.

Figure 5: Comparative study of the DNA-binding and catalytic properties of wild-type and RAL-resistant INs. (A) 3′-Processing activity—after 3 h of incubation at 37°C—as a function of IN concentration. 3′-Processing activities were quantified as described in Materials and methods section, using a 21-mer DNA substrate (4 nM), with MgCl2 as a cofactor (10 mM) in 20 mM Hepes (pH 7.2), 1 mM DTT and 30 mM NaCl. (B) DNA binding of wild-type and mutant INs. The DNA-binding step was assessed by steady-state fluorescence anisotropy as described in Materials and methods section. Experimental conditions were similar to those described in A. IN and DNA were incubated together for 15 min before recording steady-state anisotropy. (C) Kinetics of 3′-processing for the different proteins. IN concentration was 200 nM. The same symbols were used in panels A, B and C: (open square) wild-type NL-43; (filled square) G140S/Q148H NL-43; (filled triangle) Q148H NL-43; (open triangle) G140S NL-43; (open circle) wild-type patient; (filled circle) G140S/Q148H patient. Strand transfer products are depicted (middle panel). The percentages of strand transfer (shown besides the gel) were obtained after the normalization by the 3′-processing activity.

Mentions:
A plot of 3′-processing activity (corresponding to 3 h of incubation at 37°C) as a function of IN concentration gave a characteristic bell-shaped curve, with activity peaking at a concentration of about 200 nM, for both WT IN, expressed in NL-43 context (Figure 5A, left panel) or in patients context before RAL treatment (Figure 5A, right panel) in accordance with previous results (13,14). Under the same conditions, the activities of the Q148H, G140S and G140S/Q148H were severely impaired, with the degree of impairment as follows: Q148H < < G140S/Q148H ≈ G140S < WT (Figure 5). It is important to note that both proteins, WT and G140S/Q148H, in the patient background displayed stronger activity than INs in the NL-43 context. We are currently investigating the effects of the polymorphism on intrinsic IN activity.Figure 5.

Figure 5: Comparative study of the DNA-binding and catalytic properties of wild-type and RAL-resistant INs. (A) 3′-Processing activity—after 3 h of incubation at 37°C—as a function of IN concentration. 3′-Processing activities were quantified as described in Materials and methods section, using a 21-mer DNA substrate (4 nM), with MgCl2 as a cofactor (10 mM) in 20 mM Hepes (pH 7.2), 1 mM DTT and 30 mM NaCl. (B) DNA binding of wild-type and mutant INs. The DNA-binding step was assessed by steady-state fluorescence anisotropy as described in Materials and methods section. Experimental conditions were similar to those described in A. IN and DNA were incubated together for 15 min before recording steady-state anisotropy. (C) Kinetics of 3′-processing for the different proteins. IN concentration was 200 nM. The same symbols were used in panels A, B and C: (open square) wild-type NL-43; (filled square) G140S/Q148H NL-43; (filled triangle) Q148H NL-43; (open triangle) G140S NL-43; (open circle) wild-type patient; (filled circle) G140S/Q148H patient. Strand transfer products are depicted (middle panel). The percentages of strand transfer (shown besides the gel) were obtained after the normalization by the 3′-processing activity.

Mentions:
A plot of 3′-processing activity (corresponding to 3 h of incubation at 37°C) as a function of IN concentration gave a characteristic bell-shaped curve, with activity peaking at a concentration of about 200 nM, for both WT IN, expressed in NL-43 context (Figure 5A, left panel) or in patients context before RAL treatment (Figure 5A, right panel) in accordance with previous results (13,14). Under the same conditions, the activities of the Q148H, G140S and G140S/Q148H were severely impaired, with the degree of impairment as follows: Q148H < < G140S/Q148H ≈ G140S < WT (Figure 5). It is important to note that both proteins, WT and G140S/Q148H, in the patient background displayed stronger activity than INs in the NL-43 context. We are currently investigating the effects of the polymorphism on intrinsic IN activity.Figure 5.

Bottom Line:
Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway.We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation.In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context.

ABSTRACTRaltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation 'freezes' IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.